MXPA01005565A

MXPA01005565A - Substances wk-5344a and wk-5344b and process for producing the same

Info

Publication number: MXPA01005565A
Application number: MXPA/A/2001/005565A
Authority: MX
Inventors: Satoshi Omura; Hiroshi Tomoda; Yoko Takahashi
Original assignee: The Kitasato Institute
Filing date: 2001-06-04
Publication date: 2002-03-05

Abstract

Novel substances WK-5344A and WK-5344B having an effect of inhibiting cholesteryl ester transfer protein;and a process for producing the same which comprises culturing a microorganism belonging to the genus Streptomyces and being capable of producing the substances WK-5344A and WK-5344B in a medium, thus accumulating the substances WK-5344A and WK-5344B in the culture and collecting the substances WK-5344A and WK-5344B therefrom. Because of showing a potent inhibitory activity on cholesteryl ester transfer protein, these substances are useful in preventingand treating human diseases induced by the accumulation of cholesterol.

Description

SUBSTANCE WK-5344A AND SUBSTANCE WK-5344B AND PROCESS FOR THEIR PRODUCTION BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to substances WK-5344A and WK-5344B and to their production. More particularly, the present invention relates to novel substances, the substance WK-5344A and the substance WK-5344B, which have an action of inhibition of the cholesteryl ester transfer protein, and their production. 2. Description of the Related Art Several preventive drugs for myocardial infarction and cerebral accident caused by the accumulation of cholesterol in the vascular wall are known as, for e; atherosclerosis caused by hypertension in adults. These drugs include, for example, pravastatin, mevinclin and fluvastatin (Endo, A. Journal of Medicinal Chemistry 25, 401-405, 1985 and Endo. A. Journal of Lipid Research 33, 1569-1582, 1992). The onset of myocardial infarction and stroke and the process of these diseases are very complicated and complex. Therefore, substances that have different mechanisms of action for the treatment of these diseases have been urgently required. Recently, as a result of westernization of the way of life in terms of food, the cause of death due to myocardial infarction and stroke as a result of the accumulation of cholesterol in the muscle wall has increased, as well as the problem of diseases related to the lifestyle. Most cholesterol is esterified mainly in the liver and intestine by long chain fatty acids to form cholesteryl esters that are secreted in the blood as components of chylomicron and very low density lipoprotein, and circulate in the blood mainly as components of low density lipoprotein and high density lipoprotein. Low-density lipoprotein, which transfers cholesterol from the liver to peripheral tissues, is a risk factor for stimulating arteriosclerosis. On the contrary, high density lipoprotein collects cholesterol from peripheral tissues and is believed to be a factor in suppressing the progression of atherosclerosis. The cholesteryl ester transfer protein is considered to be involved in the cholesteryl ester exchange reaction in both lipoproteins, and is involved in the maturation of low density lipoprotein. Therefore, a substance that inhibits the function of the cholesteryl ester transfer protein, develops a decrease in low density lipoprotein, the risk factor for arteriosclerosis in the blood, and, contrary to this, develops an arteriosclerotic action as result of the increase in high density lipoprotein which suppresses arteriosclerosis and is an effective element to prevent these diseases. COMPENDIUM AND OBJECT OF THE INVENTION We have studied a metabolic product of microorganisms isolated from soils in order to find novel bioactive substances, and we found that the cultivated mass of microbial strain WK-5344 newly isolated from soil produced substances that have a inhibition action against cholesteryl ester transfer protein. As a result of the isolation and purification of said cholesteryl ester transfer protein inhibitor from said cultured mass, since such substances having said chemical structures were not known, we have designated these substances as substance WK-5344A and substance WK -5344B. The present invention has been achieved in accordance with said knowledge. An object of the present invention is to offer a substance WK-5344A having the following formula: Another object of the present invention is proporcic: a substance WK-5344B having the following formula: A further object of the present invention is to provide a process for the production of substance WK-5344A and substance WK-5344B or salt thereof, which comprises the cultivation of a microorganism belonging to the genus Streptomyces and having the ability to produce substance WK-5344A and substance WK-5344B, the accumulation of substance WK-5344A and substance WK-5344B in a mass cultivation and isolation of substance WK-5344A and substance WK-5344B from said cultivated mass. DESCRIPTION OF THE PREFERRED MODALITIES In accordance with the preferred embodiment of the present invention, the present invention relates to the production process of novel substance, substance WK-5344A and substance WK-5344B, or salt thereof, wherein the microorganism which belongs to the genus Streptomyces which has the ability to produce the substance WK-5344 and the substance WK-5344B is Streptomyces sp. WK-5344. The present invention further relates to a microorganism belonging to the genus Streptomyces which has the ability to produce the substance WK-5344 and the substance WK-5344B and said microorganism is Streptomyces sp WK-5344 (FERM BP-6668). The microorganism having the ability to produce the substance WK-5344A and the substance WK-5344B of the present invention (hereinafter referred to as the microorganism that produces the substance WK-5344) belongs to the genus Streptomyces, and it is sufficient that it has the ability to produce the substance WK-5344A and the substance WK-5344B without limitation. Example of the preferred microbial strain used to produce substance WK-5344A and substance WK-5344B of the present invention is Streptomyces sp WK-5344, which is isolated from soil freshly collected by the present inventors. The taxonomic properties of the present strain are the following. (I) Morphological Properties Vegetative mycelia grow well on several agar media and no fragmentation was observed. Aerial mycelia grow abundantly on inorganic salts-starch agar and giicerol-asparagine agar to present a white to gray color. In microscopic observation, micelles showing spirals and chains of more than 20 spores are observed. The shape of the spores is oval with a size of 1.0 x 0.5 μm. The surface of the spore is thorny. Sclerotium, sporangium and zoospora were not observed. (II) Culture properties in various agar media The culture properties of the present production strain observed according to the method of EB Shirling and D. Gottlieb (International Journal of Systematic Bacteriology, 16, 313, 1966) appear in the Table 1. Color shades are determined with reference to Color Harmony Manual, fourth edition (Container Corporation of America, Chicago, 1958) as a standard color, and indicated as the color tone name with its code in parentheses. The following table indicates, if not specifically defined, the results of visual observation of the culture status of this strain in various culture media at a temperature of 27 ° C for 2 weeks. Table 1 Sucrose nitrate agar Growth Well, mustard toasted color, clear (2ie) reverse Toast color of light mustard - moss green (2ie-241g) abundant aerial mycelium, ash (5fe) soluble pigment no production of glucose-Asparagine agar (ISP) Growth Well, Bamboo (2gc) Reverse Bamboo (2gc) abundant aerial mycelium, alabaster color - ash (13ba-5fe) soluble pastel yellow pigment (ldb) Glycerol-asparagine agar (ISP) Good growth, yellowish color (2ec) g) Reverse bamboo (2gc) abundant aerial mycelium, alabaster color - ash (13ba-5fe) soluble pastel yellow pigment (ldb) Inorganic salts-starch agar (ISP) Good growth, bamboo (2gc) camel back (3ie) abundant aerial mycelium, ash (5fe) Soluble pigment absence of production Tyrosine agar (ISP) Good growth, bamboo (2gc) Back color toasted light mustard - green Moss (2ie-241g) abundant aerial mycelium, alabaster (13ba) Soluble pigment without production Flour agar of oats (ISP) Moderate growth, bamboo - golden yellow (2fb-2kb) reverse bamboo - coffee (2fb-21i) moderate aerial mycelium, ash (5fe) soluble pigment yellow lemon (lgc) Agar of yeast extract-malt extract (ISP) good growth, bamboo (2gc) reverse mustard (21e) abundant aerial micelle, pearl gray (13cb) soluble pigment olive yellow (lie) Nutrient agar Moderate growth, bamboo (2gc) Gold back (2nc) Moderate aerial mycelium, ash (5fe) P soluble structure without production Peptone agar-yeast-iron extract (ISP) Moderate growth, ground-flat gold (2ng) Reverse color toasted light mustard (2ie) Poor aerial miice, white (a) Soluble pigment without production Glucose-nitrate agar ( ISP) Moderate growth, tan color light mustard (2ie) reverse mustard (21e) moderately aerial, white airy ("golden soluble pigment (1 l / 21c) Calcium glycerol-malate augmentation Moderate growth, bamboo (2gc) Reverse light wheat - bamboc (2ea-2gc) abundant aerial mycelium, pearl (3ba) soluble pigment lemon (lgc) glucose-peptone agar moderate growth, bamboo (2gc) light wheat back-golden yellow (2ea-2kb) poor aerial mike, white ( a) Soluble pigment without production (III) Physiological properties (1) Melanin pigment (a) Tyrosine negative agar (b) Peptone agar-yeast extract-negative iron (c) Glucose-peptone-negative gelatin medium (d) Tryptone broth-negative yeast extract (2) Tyrosinase negative reaction (3) Production of negative hydrogen sulphide (4) Reduction of positive nitrate (5) Gelatin liquefaction (21-23 ° C) negative (glucose-peptone-gelatin medium) (6) Hydrolysis of positive starch (7) Coagulation of skim milk (37 ° C) negative (8) Peptonization of skim milk (37 ° C) positive (9) Growth temperature 9-37 ° C (10) Utilization of carbon sources (Pridham and Gottlieb medium) Used: glucose, arabinose, xylose, melibose, mannitol, rhamnose, fructose and inositol Slightly used: raffinose and sucrose (11) decomposition of negative cellulose (IV) Cell wall composition The cell wall diaminopimelic acid is of the LL type.

The taxonomic properties of the present strain are summarized below. The diaminopimelic acid in the cell wall is of the LL type. The vegetative mycelia show good growth on several agar media and no fragmentation was observed. The shape of the aerial mycelium is spiral with long chains of spores. The surface of the spore is thorny. Several properties in culture present a brown color of vegetative mycelium and a white to gray color of aerial mycelia. Soluble pigment formation is greenish yellow on yeast extract-malt extract agar medium, oatmeal agar medium, glucose-asparagine agar medium, glycerol-asparagine agar medium, glucose agar medium -nitratc, calcium glycerol-malate agar medium. In accordance with the results of these observations, the present strain has been identified as a strain belonging to the genus Streptomyces and was known as Streptomyces sp WK-5344. The present strain was deposited with the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located at AIST Ts kuba Central 6, 1 - 1, Higashi 1-chcme Tsukuba-shi, Ibaraki-ken, 305-8566 Japan, with number FERM BP-6668 on the first day of March 1999. Although the substance WK-5344 producing a microbial strain was explained as a preferable strain of the present invention, the taxonomic properties they are very easily mutated as general properties of microorganisms and are not stable. It is also known to carry out mutations by natural mutation techniques or artificial mutation techniques such as irradiation with ultraviolet light, X-ray irradiation or application of mutagenic agents such as N-methyl-N '-nitro-N-nitrosoguanidine and methanesulfonate of ethyl. Accordingly, strains having the ability to produce a substance WK-5344 belonging to the genus Streptomyces including artificial mutants and natural mutants can be employed in the present invention. Strains mutated by cellular engineering technology such as cell fusion and gene manipulation are included within the substance production strain WK-5344. In the production of substance WK-5344A and substance WK-5344B of the present invention, the microorganism which produces substance WK-5344 belonging to the genus Streptomyces is cultured in a preferred medium. In the culture of the present microorganism, a culture method for common microorganism is generally provided. Examples of media are nutrient media containing carbon sources assimilable for microorganisms, digestible nitrogen sources for microorganisms and, if necessary, addition of inorganic salts. Examples of the assimilable carbon sources above are glucose, sucrose, molasses, starch, dextrin, cellulose, and organic acid and are used alone or in combination. Examples of sources of digestible nitrogen are a source of organic nitrogen, for example, nitrone, yeast extract, dry yeast, soybean powder, maceration corn liquor, cottonseed oil, caffeine, soy protein hydrolyzate, amino acids and urea, and inorganic nitrogen source, such as, for example, nitrate and ammonium salt, and are used alone or in combination. Further, if necessary, inorganic salt is added, for example sodium salt, potassium salt, calcium salt, magnesium and phosphate salt, and heavy metal salt. In the medium, minor nitrogen elements, substance that stimulate growth or precursor to stimulate the growth of the present strain and substance production WK-5344A and WK-5344B may be added. The culture can be carried out in an aerobic condition such as, for example, culture with agitation or culture with agitation and ventilation. The pH of the medium is preferably maintained in * a neutral condition. For industrial production, submerged ventilation is preferred. The culture condition can be carried out within a range of 20 to 37 ° C, generally at a temperature comprised within a range of 24 to 30 ° C, preferably at 27 ° C. The cultivation time is generally from 2 to 6. days in the liquid culture to accumulate the substance EK-5344 and WK-5344B and the crop can be finished when the maximum production of substance WK-5344A and substance WK-5344B can be achieved. The culture condition such as for example medium composition, culture temperature, stirring speed and ventilation volume can be adjusted and selected in order to obtain preferable conditions according to the types of strains used and the external conditions. In liquid culture, if foam formation occurs, antifoam agents such as silicon oil, vegetable oil and surfactants may be employed. Since the substance WK-5344 and substance WK-5344B accumulated in the cultivated mass are in the cultivated liquid or cultivated micelle, the cultivated mass is filtered by the addition of a filter aid such as Celite and Hyflosupercell or the cultivated mass is centrifuged to separate the culture liquid from the micelles, which are extracted with organic solvent, then the extracts are advantageously concentrated and the substance WK-5344A and the substance WK-5344B are isolated. The substance WK5344A and the substance WK-5344B are isolated from the culture filtrate by extracting the culture filtrate with an organic solvent which is immiscible with water, such as, for example, ethyl acetate, butyl acetate and benzene, and vacuum extract to obtain substance WK-5344A and substance WK-5344B. Said crude substance can be purified by the known method used for the purification of lipophilic substance. For example, column chromatography using a vehicle such as, for example, silica gel or alumina to obtain a purified substance WK-5344 and a substance WK-5344B. In order to isolate the substance WK-5344A and the substance WK-5344B from the mycelia, the micelles are extracted with a water-miscible organic solvent, aqueous, such as aqueous methanol, the extract is concentrated in a vacuum, the concentrated with an organic solvent immiscible in water such as ethyl acetate, butyl acetate, or benzene, and the extract is purified with or without a combination of the previous extract obtained from the culture liquid to obtain the substance WK-5344A and the substance WK-5344B. The physicochemical properties of the substance WK-5344A and of the substance WK-5344B of the present invention are described as follows. (I) substance WK-5344A (1) Molecular Formula: C 5 AoA 013Fe (measured in high resolution FAB mass spectrum (positive), m / z 877.1203 (M + H)) (calculated: 877.1206) (2) Molecular Weight: 876 (m / z 877 (M + H) + and 899 (N + Na) + are observed by mass spectrum (positive)) (3) Specific rotation: (a) c 2 5 -3000 ° (c = 0.01, methanol) (4) UV spectrum: UV spectrum measured in methanol is shown in Figure 1. Peak specific peaks are observed at approximately 280, 305 (shoulders), 440 and 690 nm. (5) IR spectrum: the IR spectrum (table KBr) appears in figure 2,? max '"' ~ 1 c" 1; 3400, 1728, 17C1, 1597, 1493, 1385, 1284, 1207 and 1105. (6) Solubility in solvents: Soluble in methane !, ethanol, acetonitrile, ethyl acetate, chloroform and dimethyl sulfoxide. Insoluble in water. (7) Nature of the substance: neutral (8) Color and substance form: greenish powder (9) Proton NMR: Figure 3 (Varian NMR, in deuterium methanol, 400 MHz) (10) Carbon NMR: Figure 4 ( They vary NMR, in deuterium methanol, 100 MHz) the content of the substance WK-5344A was determined considering the aforementioned physicochemical properties and spectrum data as follows I II) Substance WK-5344B (1) Molecular Formula: C. H. N. 0; Fe (in high resolution FAB mass spectrum (positive), measured m / z 905.1151 (MH) is measured) (calculated: 905.1155) (.2) Molecular Weight: 904 (m / z 905 (M + H) + and observed by mass spectrum FAB (positive)) (3) Specific rotation: (a) - -? -1000 ° (c = 0.01, methanol) (4) UV spectrum: UV spectrum measured in methanol is shown in Figure 5. Peak specific peaks are observed at approximately 285, 303 (shoulders), 445 and 698 nm. (5) IR spectrum: the IR spectrum (table KBr) appears in figure 6,? max "r cirf-; 3400, 1718, 1701, 1597, 1508, 1385, 1281, 1196 and 1105. (6) Solubility in solvent: soluble in methanol, ethanol, acetonitrile, ethyl acetate, chloroform and dimethyl sulfoxide. in water (7) Nature of the substance: neutral (8) Color and shape of the substance: greenish powder (9) Proton NMR: Figure 7 (Varian NMR, in deuterium methanol, 400 MHz) (10) Carbon NMR: Figure 8 (Varied NMR, in deuterium methanol, 100 MHz) The structure of substance WK-534 3 was determined considering the physical-chemical properties above and the spectral data that we present below.

As described above, the physico-chemical properties of substance WK-5344A and substance WK-5344B were explained in detail, no identical compounds were reported with these properties, and substance WK-5344A and substance WK- 5344B are determined as novel substances. The biological properties and the inhibitory activity of the substance WK-5344A and of the substance WK-5344B of the present invention are explained below. (1) Inhibitory action for the human cholesteryl ester transfer protein The effect of the cholesteryl ester transfer protein is determined by employing a crude protein prepared from human plasma according to the method described in Kato, et al. Journal of Biological Chemistry, 264 ^ 4082-4087, 1989. A reconstituted high-density lipoprotein (designated below as HDL) containing 25 μl of [1-A] cholesteryl ester, 10 μl of low density lipoprotein of human origin ( then LDL), 30 μl of 5 M 5,5-dithiobisnitrobenzoic acid, and 5 μl of partially purified human cholesteryl ester transfer protein were combined. A total reaction mixture of 150 μl reacted at a temperature of 37 ° C for 30 minutes.

After the reaction, 5 μl of 0.1% dextran sulfate, 5 μl of 6 mM MgCl 2 and 20 μl of phosphate buffer adjusted to an ionic strength of 0.16 were added, and the mixture was left on ice for 20 minutes. The mixture was centrifuged at a temperature of 4 ° C, 13000 rpm for 15 minutes. The LDL fraction of precipitate was collected. LDL was dissolved in 180 μl of 0.1 N NaOH, and the cholesteryl ester transferred into LDL was measured by liquid scintillation counting. The results of 50% inhibition of cholesteryl ester transfer protein activity were 0.54 μg / ml substance WK-5344A and 2.0 μg / ml substance WK-5344B. As described above, the substance WK-5344A and the substance WK-5344B of the present invention show a significant inhibitory action against the cholesteryl ester transfer protein and are considered to be useful for the prevention and treatment of diseases caused by cholesterol accumulation in humans. BRIEF EXPLANATION OF THE DRAWINGS Figure 1 shows the UV spectrum of the substance WK-5344A of the present invention (in CH 3 OH). Figure 2 shows the IR spectrum of substance WK-5344A of the present invention (KBr tablet). Figure 3 shows an NMR spectrum of protons of substance WK-5344A of the present invention (in CD ^ OD).

Figure 4 shows carbon NMR spectrum of substance WK-5344A of the present invention (in CD3OD). Figure 5 shows the UV spectrum of substance WK-5344B of the present invention (in CH 3 OH). Figure 6 shows the IR spectrum of substance WK-5344B of the present invention (KBr tablet). Figure 7 shows an NMR spectrum of protons of substance WK-5344B of the present invention (in CD3OD). Figure 8 shows a carbon NMR spectrum of substance WK-5344B of the present invention (in CD ^ OD). DETAILED DESCRIPTION OF MODALITIES OF THE INVENTION The following example illustrates the present invention but does not limit the present invention. Example 100 ml of medium (adjusted to pH 7.0) containing 2.4% starch, 0.5% yeast extract, 0.1% glucose, 0.3% peptone, 0.3% meat extract and 0.4% CaCO3 was sterilized in steam. in a 500 ml Erlenmeyer flask, sealed with a cotton plug. After cooling, a platinum loop of Streptomyces sp. Was inoculated aseptically there. WK-5344 FERM BP-6668 grown on an agar medium and cultivated with agitation at a temperature of 27 ° C for 72 hours to obtain a seed culture liquid. A medium (adjusted to a pH of 6.5) containing 4.0% soluble starch, 2.0% roasted soybean meal extracted with solvent, 32 μl / 1 0.1N sodium thiosulfate, 0.05% FeSO < • 7H20, 0.05% KH2P04 and 0.03% KCl in a 30 liter fermenter was steam sterilized. After cooling, 200 ml of seed culture liquid were aseptically inoculated, and cultured with shaking at 250 rpm, ventilating 10 liters / minute at 27 ° C for 96 hours. After cultivation, the mycelia obtained by centrifugation of the cultivated liquid were extracted with 6 liters of acetone. The extract was concentrated in vacuo to remove acetone, adjusted to pH 5.0 extracted with 10 liters of ethyl acetate and the extract was concentrated in vacuo to obtain 9.20 g of crude substance. The crude substance was divided into hexane, methanol and water (40: 19: 1), substance WK-5344 was collected, which was divided in the lower layer, and concentrated in vacuo to obtain 1.29 g of crude substance. The crude substance was suspended in 10 ml of acetonitrile, loaded on ODS column (20 ml, Senshu Co., SSC-ODS-7515-12), and chromatographed with elution with acetonitrile containing 0.05% phosphoric acid. A fraction of 12 ml of eluate was collected, and the fractions containing the active ingredient were collected. After removal of the acetonitrile, the aqueous layer fraction was extracted with ethyl acetate and dried in vacuo to obtain 157 mg of crude active substance. The 157 mg of raw active substance were dissolved in 1.57. ml of methanol, loaded on an ODS column (147 ml, Senshu Co., SSC-ODS-7515-12), and chromatographed stepwise with acetonitrile containing 0.05% phosphoric acid. 12 ml of the eluted product fraction were collected and the fractions containing the active ingredient were collected. After removal of the acetonitrile, the aqueous layer fraction was extracted with ethyl acetate and dried in vacuo to obtain 19.5 mg of crude active substance. The 19.5 mg of raw active substance were dissolved in 0.5 ml of chloroform, loaded on a column of silica gel (126 ml, silica gel 60), and chromatographed with elution with chloroform and methanol. Each fraction was fractioned with 12 mi. As a result, 1.69 mg of substance WK-5344A and 1.12 mg of substance WK-5344B were isolated. EFFECT OF THE INVENTION In accordance with what has been described above, a microorganism belonging to the genus Streptomyces was cultivated which has the ability to produce substance WK-5344A and substance WK-5344B in a medium to accumulate the substance WK-5344A and the substance WK-5344B in the cultivated mass. The substance WK-5344A and the substance WK-5344B were collected from said cultivated mass to obtain substances having an action of inhibition on the cholesteryl ester transfer protein. It is expected that said substances show preventive and curative effects for adult diseases such as myocardial infarction and cerebral accidents caused by arterial sclerosis.

Claims

CLAIMS A substance WK-5344A represented by the following formula or a salt of it. A substance WK-5344B represented by the following formula or a salt of it. A process for the production of substance WK-5344A and substance WK-5344B or a pharmaceutically acceptable salt thereof, comprising the culture of a microorganism belonging to the genus Streptomyces and having the ability to produce the substance WK-5344A and the substance WK-5344B in a medium, accumulate substance WK-5344A and substance WK-5344B in a cultivated medium, and isolate substance WK-5344A and substance WK-5344B from the cultivated medium, and if required, substances are converted to a pharmaceutically acceptable salt thereof. The process according to claim 3, wherein the microorganism belonging to the genus Streptomyces and having the ability to produce the substance WK-5344A and the substance WK-5344B is Streptomyces sp. WK-5344. A microorganism that belongs to the genus Streptomyces and that has the ability to produce a substance WK-5344A and a substance WK-5344B. The microorganism according to claim 5, wherein the microorganism is Streptomyces sp. WK-5344 FERM BP-6668.